| One third of the human lifetime is spent in sleep.Sleep and wakefulness display the different functional states of the brain,and are divided into wakefulness（W）,rapid eye movement（REM）sleep and non-REM（NREM）sleep according to the polysomnographic criteria by recording electroencephalogram（EEG）and electromyogram（EMG）.The cotrol mechanism of sleep-wake cycle in the wildely accepted transition model of sleep-wake states shows that the γ-aminobutyric acid（GABA）neurons in the ventrolateral preoptic nucleus（VLPO）of hypothalamus that promote sleep project to and interact with the tuberomammillary nucleus（TMN）hitamine（HA）,the lateral hypothalamus（LH）orexin（Orx）,the brainstem noradrenalin（NA）and serotonin（5-HT）neurons that promote wakefulness to modulate sleep-wake cycle.Drugs that excite GABAergic neurons or inhibit HAergic or Orxergic neurons are clinically used to treat insomnia,but have been shown to have several varieties of adverse and side effects.The methods of non-drug treating insomnia are more and more demanded for clinical practice.Acupuncture in the Traditional Chinese Medicine（TCM）have been used to treat insomnia for more than thousand year in Chinese history.Shenmen（HT7）and Sanyinjiao（SP6）acupoints are most frequently used for insomina therapy.It is well known that the acupuncture skills and acupoints used in every Doctor’s treatment,and the outcome and efficacy in each patient are variouse,so the method of acupuncture for insomina therapy is not widely acceptable in the world.Therefore,it is necessary to use polysomnography to objectively evaluate the effects of acupuncture at single and multi acupoints on sleep.Electroacupuncture（EA）,a simulating artificial acupuncture,is able to control the frequency and intensity of stimulating pulse,and is easily and widely popularized for acupuncture therapy.In this study,we desiged to use the adult male rats as experimental subjects and record their EEG and EMG signals in freedom for analyzing the effect of EA stimulation with various pulse frequencies and intensities at HT7,SP6 and HT7+SP6 acupoints on the sleep-wake states.c-Fos,an immediate early gene,was employed to find the active neurons in the brain using immunohistochemistry following the EA stimulation that induced best promoting sleep for revealing the potential targets and the underlying mechanism.The aim of the study is to provide preclinical experimental evidences for acupuncture treating insomnia.Methods: Adult male Sprague-Dawley rats under anesthesia were implanted with acupuncture needles respectively at both sides of HT7 and SP6 acupoints in EA and non-EA（NEA）stimulation groups,or 10 mm above the corresponding acupoints in non-acupoint（NAP）stimulation group.Two pairs of EEG electrodes were respectively implanted into frontal and parietal skull above the dura for recording the neocortical EEG,and three EMG electrodes were implanted into nuchal muscles for recording EMG.The electrodes are connected to a miniature socket and fixed to the skull surface for connecting the recording cable.One week after recovery,the following experiments were carried out: Experiment 1: A 12-h sleep-wake cycle was recorded after a EA with 0.3 m A,20 Hz,pulse width 0.2 ms for 20 min stimulated the animals listed as follows: 1)Shenmen acupoint（HT7）,rats were divided into（1）NEA group,non-EA stimulation though acupuncture needle implanted,n=7;（2）NAP group,EA stimulation was at 10 mm above HT7 acupoint,n=7;（3）HT7 group,EA stimulation delivered at HT7 acupoint through an implatation acupuncture needles,n=7.2)Sanyinjiao acupoint（SP6）,grouping as（1）NEA group,n=7;（2）NAP group,n=7;（3）SP6 group,n=7.3)Shenmen plus Sanyinjiao acupoints（HT7+SP6）,rats were divided into（1）NEA group,n=7;（2）NAP group,n=7;（3）HT7+SP6 group,n=7.Experiment 2.Based on the results of experiment 1,EA stimulation at HT7+SP6 was better than any single acupoint in the efficacy of promoting sleep.The following experiments were further carried out: 1)EA at HT7+SP6 with 0.3 m A,100 Hz,pulse width 0.2 ms for 20 min,and then a 12-h sleep-wake cycle was recorded.Animal groups:（1）NAP group,n=7;（2）HT7+SP6 group,n=7.2)EA at HT7+SP6 with 0.3 m A,alternate cycles of 20 Hz 5 s and 100 Hz 10 s,pulse width 0.2 ms for 20 min,and then a 12-h sleep-wake cycle was recorded.Animal groups:（1）NAP group,n=7;（2）HT7+SP6 group,n=7.3)EA at HT7+SP6 with 1.5 m A,20 Hz,pulse width 0.2 ms for 20 min,and then a 12-h sleep-wake cycle was recorded.Animal groups:（1）NAP group,n=7;（2）HT7+SP6 group,n=7.Experiment 3.The neurons in the brain were labeled with c-fos using immunohistochemistry following EA with 1.5 m A,20 Hz,pulse width 0.2 ms for 20 min stimulation at HT7+SP6.Results: 1.EA（0.3 m A,20 Hz）stimulation at NAP showed undifferentiated from NEA group in the hourly and total amount of 12-h sleep-wake states,while the same stimulation at HT7,SP6 or HT7+SP6 respectively increased the 12-h NREM sleep amount by 28.2%（p < 0.001）,19.7%（p < 0.01）and 48.9%（p < 0.001）compared with the NAP group.However,there was no statistical difference in REM sleep between NEA group and all EA groups.2.The 12-h NREM sleep amount induced by EA（0.3 m A,20 Hz）stimulation at HT7+SP6 was respectively 10.6%（p < 0.01）and 24.7%（p < 0.01）more than that induced by the same stimulation at HT7 and SP6,as well 21.2%（p < 0.001）and 10.4%（p < 0.01）more than that induced respectively by EA（0.3 m A,100 Hz）and EA alternate cycles（20 Hz,5 s and 100 Hz,10 s）stimulation at HT7+SP6.3.When EA（1.5 m A,20 Hz）stimulation delivered at HT7+SP6,the 12-h NREM sleep amount was increased by 58.3%（p < 0.001）compared to NAP group,and by 5.5% compared to EA（0.3 m A,20 Hz）stimulation at HT7+SP6 also,indicating that EA（1.5 m A,20 Hz）stimulation at HT7+SP6 execute the best effect on promoting sleep.The analysis of sleep architecture demonstrated that the increased NREM sleep induced by EA（1.5 m A,20 Hz）stimulation at HT7+SP6 was through reducing sleep latency,increasing the episode number and mean duration of NREM sleep.4.EA（1.5 m A,20 Hz）stimulation at HT7+SP6 increased the number of Fos-ir neurons in the VLPO by 193.1%（p < 0.001）compared with NAP group.Conclusion: EA（0.3 m A,20 Hz）stimulation at HT7 or SP6 promotes NREM sleep,while the amount of increased NREM sleep in the HT7+SP6 acupoints is more than that in any single acupoint.Notably,when the EA intensity is increased,the EA（1.5 m A,20 Hz）stimulation at HT7+SP6 induce the best effect on promoting NREM sleep.This effect is mainly achieved by reducing sleep latency,increasing the episode number and mean duration of NREM sleep.Furthermore,EA（1.5 m A,20 Hz）stimulation at HT7+SP6 enhances neuronal activity of the VLPO,suggesting that the acupuncture-induved promoting sleep might be through the enhancement of GABAergic neuronal activity in the VLPO and the inhibition of arousal neuronal activity. |
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